Quorum sensing (QS) is an emerging research field in health and environmental sciences during past decade. QS is the ability of bacteria to communicate and coordinate behavior such as biofilm formation via signaling molecules called autoinducers (e.g. acyl-homoserine lactones (AHLs)), and is now known to be involved in many microbial disease and biofouling processes. Therefore, it is conceivable that quorum sensing inhibition represents a new, novel strategy to disrupt biofilm formation to combat microbial diseases.
A simple, sensitive and rapid assay for the detection of AHLs is urgently needed in quorum sensing research. There are many methods developed to detect AHLs, such as whole cell assays using biosensors, thin-layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), and liquid chromatography/mass spectrometry (LC/MS). Among those methods, whole-cell bioassays, such as the reporter bacterium Agrobacterium tumefaciens NTL4 (pCF218)(pCF372), have been a most popular and sensitive method because these methods don't require expensive instruments (such as in HPLC and LC/MS). However, there are disadvantages of the whole-cell bioassay: 1) it requires relatively labor intensive cell conditioning, 2) it takes at least 24 hours for detection of AHLs, 3) it may be difficult to screen the inhibitors of QS when the test substance inhibits cell growth itself and 4) it is qualitative and does not provide accurate and precise quantification.
The in-vitro synthesis of proteins in cell-lysate extracts has been an important tool for molecular biologists and has a variety of applications including rapid identification of gene products (e.g. proteomics), localization of mutations through synthesis of truncated gene products, protein folding studies, and incorporation of modified or unnatural amino acids for functional studies. The most frequently-used cell-free translation systems consist of extracts from rabbit reticulocycles, wheat germ and Escherichia coli. All are prepared as crude extracts containing all the macromolecular components (e.g. 705 or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA.
As such, a need exists for an improved method of sensing AHLs. The present disclosure seeks to address the disadvantages of prior art construction and methods, and presents a high-throughput in-vitro translation (cell-lysate based) assay system for detection of QS chemical signals.